This invention relates to relatively short peptides, and more particularly to peptides between about 16 and about 46 residues in length, which are naturally available in minute amounts in the venom of the cone snails and which may include one or more cyclizing disulfide linkages.

Background of the Invention Mollusks of the genus Conus produce a highly toxic venom which enables them to carry out their unique predatory lifestyle. Prey are immobilized by the venom which is injected by means of a highly specialized venom apparatus, a disposable hollow tooth which functions both in the manner of a harpoon and a hypodermic needle. Few interactions between organisms are more striking than those between a venomous animal and its envenomated victim. Venom may be used as a primary weapon to capture prey or as a defense mechanism. These venoms disrupt essential organ systems in the envenomated animal, and many of these venoms contain molecules directed to receptors and ion channels of neuromuscular systems.

The predatory cone snails (Conus) have developed a unique biological strategy. Their venom contains relatively small peptides that are targeted to various neuromuscular receptors and may be equivalent in their pharmacological diversity to the alkaloids of plants or secondary metabolites of microorganisms. Many of these peptides are among the smallest nucleic acid-encoded translation products having defined conformations, and as such they are somewhat unusual because peptides in this size range normally equilibrate

among many conformations for proteins having a fixed conformation are generally much larger.

The cone snails that produce these toxic peptides, which are generally referred to as conotoxins or conotoxin peptides, are a large genus of venomous gastropods comprising approximately 500 species. All cone snail species are predators that inject venom to capture prey, and the spectrum of animals that the genus as a whole can envenomate is broad. A wide variety of hunting strategies are used; however, every Conus species uses fundamentally the same basic pattern of envenomation.

The major paralytic peptides in these fish- hunting cone venoms were the first to be identified and characterized. In C. geographus venom, three classes of disulfide-rich peptides were found: the α-conotoxins (which target and block the nicotinic acetylcholine receptors) ; the μ-conotoxins (which target and block the skeletal muscle Na ⁺ channels) ; and the to-conotoxins (which target and block the presynaptic neuronal Ca ²⁺ channels) . However, there are multiple homologs in each toxin class; for example, at least five different Z-conotoxins are present in C. geographus venom alone. Considerable variation in sequence is evident, and when different fo- conotoxin sequences were first compared, only the cysteine residues that are involved in disulfide bonding and one glycine residue were found to be invariant. Another class of conotoxins found in C. geographus venom is that referred to as the conantokins which cause sleep in young mice and hyperactivity in older mice and are targeted to the NMDA receptor. Each cone venom appears to have its own distinctive group or signature of different conotoxin sequences.

Many of these peptides have now become fairly standard research tools in neuroscience. The μ- conotoxins, because of their ability to preferentially

block muscle but not axonal Na* channels, are convenient tools for immobilizing skeletal muscle without affecting axonal or synaptic events. The fo-conotoxins have become standard pharmacological reagents for investigating voltage-sensitive Ca ²⁺ channels and are used to block presynaptic termini and neurotransmitter release. The ύ- conotoxin GVIA from C. geographus venom, which binds to neuronal voltage-sensitive Ca ²⁺ channels, is an example of such. The affinity (K _d ) of Zb-conotoxin GVIA for its high- affinity targets is sub-pico olar; it takes more than 7 hours for 50% of the peptide to dissociate. Thus the peptide can be used to block synaptic transmission virtually irreversibly because it inhibits presynaptic Ca ²⁺ channels. However, Zb-conotoxin is highly tissue- specific. In contrast to the standard Ca ²⁺ channel- blocking drugs (e.g. the dihydropyridines, such as nifedipene and nitrendipene, which are widely used for angina and cardiac problems) , which can bind Ca ²⁺ channels in smooth, skeletal, and cardiac muscle as well as neuronal tissue, Zb-conotoxins generally bind only to a subset of neuronal Ca ²⁺ channels, primarily of the N subtype. The discrimination ratio for Zb-conotoxin binding to voltage-sensitive Ca ²⁺ channels in neuronal versus nonneuronal tissue (e.g. skeletal or cardiac muscle) is greater than 10 ⁸ in many cases.

Additional conotoxin peptides having these general properties continue to be sought.

Summary of the Invention The present invention provides a group of bioactive conotoxin peptides which are extremely potent inhibitors of synaptic transmission at the neuromuscular junction and/or which are targeted to specific ion channels. They are useful as pesticides, and many of them or closely related analogs thereof are targeted to specific insects or other pests. Therefore, the DNA

encoding such conotoxin peptides can be advantageously incorporated into plants as a plant-defense gene to render plants resistant to specified pests.

These conotoxin peptides have the formulae set forth hereinafter. Moreover, examination of the formulae shows an indication of two new classes of conotoxin peptides in addition to those classes hereinbefore described. Class A includes peptides SEQ ID NO:l to NO:6; each has 6 Cys residues which are interconnected by 3 disulfide linkages, with the 2 Cys residues nearest the N-terminus being part of a sequence -Cys-Cys-Gly-. All 6 members have at least one 4Hyp residue and a C-terminus which appears to be amidated. There are 2 amino acid(AA) residues separating the 3rd and 4th Cys as numbered (from the N-terminus) and a single AA residue spacing the 4th Cys from the 5th Cys. Moreover, the 2nd Cys is usually separated from the 3rd Cys by either 6 or 7 AA residues in this class, whereas there can be from about 3 to about 6 AA residues separating the 5th and 6th Cys residues. Class B is exemplified by SEQ ID NO:7, wherein there is a central sequence of 5 AA residues having 2 pairs of Cys residues flanking a center residue which is preferably Asn and wherein there are 2 additional pairs of spaced- apart Cys residues located, respectively, N-terminally and C-terminally of this central sequence. SEQ ID NO:8 appears to be a member of the known class of a- conotoxins. SEQ ID NO:9 appears to be a member of the known class of μ-conotoxins. SEQ ID NO:10 and NO:11 may be members of the class of Zo-conotoxins. SEQ ID NO:12 appears to be a member of the class of conantokins characterized by the N-terminal sequence Gly-Glu-Gla-Gla, and SEQ ID NO:13 may be a member of a heretofore uncharacterized class which causes sluggish behavior. The individual formulae of these conotoxins are as follows:

Gly-Cys-Cys-Gly-Ser-Tyr-Pro-Asn-Ala-Ala-Cys-His-Pro-Cys- Ser-Cys-Lys-Aεp-Arg-Xaa-Ser-Tyr-Cys-Gly-Gln (SEQ ID NO:l) (J-020) , wherein Xaa is 4Hyp (4-hydroxyproline) and the C-terminus is amidated;

Glu-Lys-Ser-Leu-Val-Pro-Ser-Val-Ile-Thr-Thr-Cys-Cys-Gly- Tyr-Asp-Xaa-Gly-Thr-Met-Cys-Xaa-Xaa-Cys-Arg-Cys-Thr-Asn- Ser-Cys (SEQ ID NO:2) (J-005) wherein Glu in the l- position is pGlu, Xaa is 4Hyp and the C-terminus is amidated; Ser in the 7-position may be glycosylated;

Cys-Cyε-Gly-Val-Xaa-Asn-Ala-Ala-Cys-Pro-Xaa-Cys-Val-Cys- Asn-Lys-Thr-Cys-Gly (SEQ ID NO:3) (OB-34) wherein Xaa is 4Hyp and the C-terminus is amidated;

Gly-Cys-Cys-Gly-Ser-Tyr-Xaa-Asn-Ala-Ala-Cys-His-Xaa-Cys- Ser-Cys-Lys-Asp-Arg-Xaa-Ser-Tyr-Cys-Gly-Gln (SEQ ID NO:4) (J-019) wherein Xaa is 4Hyp and the C-terminus is amidated;

Gly-Cys-Cys-Gly-Ser-Tyr-Xaa-Asn-Ala-Ala-Cys-His-Pro-Cys- Ser-Cys-Lys-Asp-Arg-Xaa-Ser-Tyr-Cys-Gly-Gln (SEQ ID NO:5) (J-026) wherein Xaa is 4Hyp and the C-terminus is amidated;

Cys-Cys-Gly-Val-Xaa-Asn-Ala-Ala-Cys-His-Xaa-Cys-Val-Cys- Lys-Asn-Thr-Cyε (SEQ ID NO:6) (OB-26) wherein Xaa is 4Hyp and the C-terminus is amidated;

Gly-Xaa-Ser-Phe-Cyε-Lys-Ala-Asp-Glu-Lyε-Xaa-Cys-Glu-Tyr - His-Ala-Aεp-Cys-Cys-Asn-Cyε-Cys-Leu-Ser-Gly-Ile-Cys-Ala- Xaa-Ser-Thr-Aεn-Trp-Ile-Leu-Pro-Gly-Cyε-Ser-Thr-Ser-Ser- Phe-Phe-Lys-Ile (SEQ ID NO:7) (J-029) wherein Xaa is 4Hyp; the C-terminus may optionally be amidated;

Gly-Cys-Cys-Ser-His-Pro-Ala-Cys-Ser-Gly-Lys-Tyr-Gln-Xaa- Tyr-Cys-Arg-Xaa-Ser (SEQ ID NO:8) (OB-20) wherein Xaa is Gla and the C-terminus is amidated;

His-Xaa-Xaa-Cys-Cys-Leu-Tyr-Gly-Lys-Cys-Arg-Arg-Tyr-Xaa- Gly-Cyε-Ser-Ser-Ala-Ser-Cys-Cys-Gln (SEQ ID NO:9) (J-021) wherein Xaa is 4Hyp;

Cys-Lys-Thr-Tyr-Ser-Lys-Tyr-Cys-Xaa-Ala-Asp-Ser-Xaa-Cys- Cys-Thr-Xaa-Gln-Cys-Val-Arg-Ser-Tyr-Cys-Thr-Leu-Phe (SEQ ID NO:10) (J-010) wherein Xaa is Gla and the C-terminus is amidated;

Ser-Thr-Ser-Cyε-Met-Glu-Ala-Gly-Ser-Tyr-Cyε-Gly-Ser-Thr - Thr-Arg-Ile-Cys-Cys-Gly-Tyr-Cys-Ala-Tyr-Phe-Gly-Lys-Lys- Cyε-Ile-Asp-Tyr-Pro-Ser-Asn (SEQ ID NO:11) (J-008) ;

Gly-Glu-Xaa-Xaa-Val-Ala-Lyε-Met-Ala-Ala-Xaa-Leu-Ala-Arg- Xaa-Aεn-Ile-Ala-Lys-Gly-Cys-Lys-Val-Asn-Cyε-Tyr-Pro (SEQ ID NO:12) (J-017) wherein Xaa is Gla (γ-carboxyglutmate) ; and

Glu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr- Ile-Leu (SEQ ID NO:13) (J-004) , wherein Glu in the 1- position is pGlu (pyroglutamic) and the C-terminus may be amidated; Thr may be glycosylated.

Accordingly in one aspect, the invention provides conotoxin peptides having the general formula: Xaa ₁ -Cys-Cys-Gly-Xaa ₂ -Cys-Xaa ₃ -Xaa ₄ -Cys-Xaa ₅ -Cys-Xaa ₆ -Cys- Xaa ₇ -NH ₂ (SEQ ID NO:14) wherein Xaa ₁ is des-Xaa ₁ or Gly or pGlu-Lys-Ser-Leu-Val-Pro-Ser-Val-Ile-Thr-Thr; Xaa ₂ is Ser-Tyr-Pro-Asn-Ala-Ala or Tyr-Asp-4Hyp-Gly-Thr-Met or Val-4Hyp-Asn-Ala-Ala or Ser-Tyr-4Hyp-Asn-Ala-Ala; Xaa ₃ is His, 4Hyp or Pro; Xaa ₄ is Pro or 4Hyp; Xaa ₅ is Ser, Arg or Val; Xaa ₆ is Lys-Asp-Arg-4Hyp-Ser-Tyr or Thr-Asn-Ser or

Asn-Lys-Thr or Lyε-Aεn-Thr; and Xaa ₇ iε deε-Xaa ₇ or Gly or Gly-Gln.

In another aspect, the invention provides conotoxin peptides having 6 Cys residues interconnected by 3 disulfide bonds, with the 2 Cys residues nearest the N-terminus being part of the sequence Cys-Cys-Gly and with the 3rd, 4th and 5th reεidue being spaced apart by 2 residues and 1 residue, respectively, said two residues being selected from His, Pro and 4Hyp, said single residue being Ser, Arg or Val and with the C-terminus being amidated, said conotoxin binding to the acetylcholine receptor.

In yet another aspect, the invention provides conotoxin peptides having 8 Cys residues interconnected by 4 disulfide bonds with the central 4 Cys reεidueε being part of the εequence Cys-Cys-Aεn-Cys-Cys (SEQ ID NO:15), said conotoxin causing immediate paralysis when administered intercranially to laboratory mice.

These peptides, which are generally termed conotoxins, are sufficiently εmall to be chemically synthesized. General chemical syntheεes for preparing the foregoing conotoxins are described hereinafter along with specific chemical syntheses of several conotoxins and indications of biological activities of these synthetic products. Various of these conotoxins can also be obtained by iεolation and purification from specific conuε species uεing the technique described in U.S. Patent No. 4,447,356 (May 8, 1984), the discloεure of which iε incorporated herein by reference. Many of these conotoxin peptides are extremely potent inhibitors of synaptic transmisεion at the neuromuεcular junction, while at the same time lacking demonstrable inhibition of either nerve or muscle action potential propagation. They are considered useful to relax certain muscleε during εurgery.

The activity of each of these conotoxin peptides is freely reversible upon dilution or removal of the toxin from the affected muscle. Moreover, toxicity of the cyclic peptides is generally deεtroyed by agentε which diεrupt diεulfide bonds in the cyclic conotoxins, suggesting that correct disulfide bonding appears essential for biological activity; however, correct folding and/or rearrangement of a conotoxin may occur in vivo so that in some cases the linear peptide may be administered for certain purposes. In general, however, the synthetic linear peptides fold spontaneouεly when expoεed to air-oxidation at cold room temperatureε to create the correct diεulfide bondε to confer biological activity, and εuch processing is accordingly preferred. The conotoxins exhibit activity on a wide range of vertebrate animals, including humans, and on insectε, and many are uεeful to reversibly immobilize a muscle or group of muscleε in humanε or other vertebrate εpecieε. Many of theεe conotoxinε and derivatives thereof are further useful for detection and measurement of acetylcholine receptors and other specific receptors which are enumerated hereinafter with respect to various particular peptides.

Many of these conotoxin peptides are also useful in medical diagnosis. For example, an immunoprecipitation asεay with radiolabeled Zb-conotoxin can be used to diagnose the Lambert-Eaton myasthenic syndrome, which is a diseaεe in which autoimmune antibodies targeted to endogenous Ca ²⁺ channels are inappropriately elicited, thereby cauεing muεcle weakness and autonomic dysfunction.

Various of these conotoxin peptides are further uεeful for the treatment of neuromuscular disorderε and for rapid reverεible immobilization of muεcleε in vertebrate εpecies, including humanε, thereby facilitating the setting of fractures and dislocations.

Theεe conotoxins generally inhibit synaptic transmiεεion at the neuromuεcular junction and bond strongly to the acetylcholine receptor of the muscle end plate, and many are therefore especially suitable for detection and assay of acetylcholine receptors. Such measurements are of particular significance in clinical diagnosis of myasthenia graviε, and various of these conotoxins, when synthesized with a radioactive label or as a fluorescent derivative, provide improved quantitation and sensitivity in acetylcholine receptor assays.

Detailed Description of the Preferred Embodiments Although the conotoxins can be obtained by purification from the enumerated cone snails, because the amounts of conotoxins obtainable from individual snails are very small, the desired substantially pure conotoxins are best practically obtained in commercially valuable amountε by chemical εyntheεis. For example, the yield from a single cone snail may be about 10 micrograms or less of conotoxin. By subεtantially pure iε meant that the peptide iε present in the subεtantial abεence of other biological moleculeε of the εame type; it is preferably present in an amount at leaεt about 85% by weight and more preferably at leaεt about 95% of such biological molecules of the same type which are present, i.e. water, buffers and innocuous small molecules may be present. Chemical synthesiε of biologically active conotoxin peptide dependε of course upon correct determination of the amino acid sequence, and these sequences have now been determined and are set forth in the preceding summary.

Many of the conotoxins have approximately the same level of activity, and comparison of them suggests a reasonable tolerance for substitution near the carboxy terminus of these peptides. Accordingly, equivalent molecules can be created by the εubstitution of

equivalent residueε in thiε region, and εuch substitutions are useful to create particular invertebrate-specific conotoxins.

Cysteine residues are present in a majority of these conotoxins, and several of the conotoxins disclosed herein exhibit similar disulfide cross-linking patterns to that of erabutoxin, a known protein toxin of sea snake venom. The fact that biological activity of these particular compounds is destroyed by agents which break disulfide bonds, such as εodium borohydride or β- mercaptoethanol, indicateε that a specific folded configuration induced by disulfide cross-links, is essential for bioactivity of these particular conotoxins. It has been found that air-oxidation of the linear peptides for prolonged periods under cold room temperatures results in the creation of a substantial amount of the bioactive, disulfide-linked molecules. Therefore, the preferable procedure for making these peptides iε to oxidize the linear peptide and then fractionate the reεulting product, uεing reverεe-phaεe high performance liquid chromatography (HPLC) or the like, to εeparate peptideε having different linked configurationε. Thereafter, either by comparing these fractions with the elution of the native material or by using a simple assay, the particular fraction having the correct linkage for maximum biological potency is easily determined. It is also found that the linear peptide, or the oxidized product having more than one fraction, can sometimes be used for in vivo administration, because the cross-linking and/or rearrangement which occurs in vivo has been found to create the biologically potent conotoxin molecule; however, because of the dilution resulting from the presence of other fractions of less biopotency, a somewhat higher dosage may be required. These conotoxins discloεed herein generally inhibit εynaptic transmission at the neuromuscular

junction by binding the acetylcholine receptor at a muscle end plate. A particularly useful characteristic of a number of these conotoxinε iε their high affinity for particular macromolecular receptorε, accompanied by a narrow receptor-target εpecificity. A major problem in medicine reεultε from εide effectε which drugε very often exhibit, some of which are caused by the drug binding not only to the particular receptor subtype that renders therapeutic value, but also to closely related, therapeutically irrelevant receptor subtypes which can often cause undesirable phyεiological effects. In contrast to most drugs, these conotoxins generally discriminate among closely related receptor subtypes. The peptides are synthesized by a suitable method, such as by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation or by clasεical solution couplings. The employment of recently developed recombinant DNA techniques may be used to prepare these peptides, particularly the longer ones containing only natural amino acid residueε which do not require poεt-translational processing steps.

In conventional solution phase peptide synthesis, the peptide chain can be prepared by a serieε of coupling reactionε in which the conεtituent amino acidε are added to the growing peptide chain in the desired sequence. The use of various N-protecting groups, various coupling reagents, e.g., dicyclohexylcarbodiimide or carbonyldimidazole, various active esterε, e.g., eεters of N-hydroxypthalimide or N- hydroxy-succinimide, and the variouε cleavage reagents, to carry out reaction in solution, with εubεequent isolation and purification of intermediates, is well known clasεical peptide methodology. Claεεical solution synthesis is described in detail in the treatise "Methoden der Organischen Chemie (Houben- eyl) : Synthese von Peptiden", E. Wunsch (editor) (1974) Georg Thieme

Verlag, Stuttgart, W. Ger. Techniques of exclusively solid-phase syntheεiε are set forth in the textbook "Solid-Phase Peptide Synthesiε", Stewart & Young, Freeman & Co., San Francisco, 1969, and are exemplified by the disclosure of U.S. Patent No. 4,105,603, issued August 8, 1978 to Vale et al. The fragment condensation method of syntheεiε iε exemplified in U.S. Patent No. 3,972,859 (Auguεt 3, 1976). Other available εyntheεeε are exemplified by U.S. Patent No. 3,842,067 (October 15, 1974) and U.S. Patent No. 3,862,925 (January 28, 1975). Common to εuch chemical εyntheεeε is the protection of the labile side chain groups of the various amino acid moieties with suitable protecting groups which will prevent a chemical reaction from occurring at that site until the group is ultimately removed. Usually also common is the protection of an alpha-amino group on an amino acid or a fragment while that entity reacts at the carboxyl group, followed by the selective removal of the alpha-amino protecting group to allow subsequent reaction to take place at that location. Accordingly, it is common that, as a step in such a εyntheεiε, an intermediate compound iε produced which includeε each of the amino acid residues located in its desired sequence in the peptide chain with appropriate side-chain protecting groups linked to various of the residues having labile side chains.

Aε far aε the selection of a side chain amino protecting group is concerned, generally one is chosen which is not removed during deprotection of the α-amino groups during the εyntheεiε. However, for εome amino acids, e.g. His, protection is not generally neceεεary. In εelecting a particular εide chain protecting group to be uεed in the εyntheεiε of the peptides, the following general rules are followed: (a) the protecting group preferably retains its protecting properties and is not split off under coupling conditions, (b) the protecting

group εhould be εtable under the reaction conditionε selected for removing the α-amino protecting group at each step of the synthesis, and (c) the side chain protecting group must be removable, upon the completion of the synthesiε containing the deεired amino acid sequence, under reaction conditions that will not undesirably alter the peptide chain.

It should be posεible to prepare many, or even all, of theεe peptideε uεing recombinant DNA technology; however, when peptideε are not εo prepared, they are preferably prepared using the Merrifield solid phase synthesis, although other equivalent chemical syntheses known in the art can also be uεed as previously mentioned. Solid-phase synthesis is commenced from the C-terminus of the peptide by coupling a protected α-amino acid to a εuitable resin. Such a starting material can be prepared by attaching an α-amino-protected amino acid by an ester linkage to a chloromethylated resin or a hydroxymethyl resin, or by an amide bond to a benzhydrylamine (BHA) reεin or paramethylbenzhydrylamine (MBHA) reεin. The preparation of the hydroxymethyl resin is described by Bodansky et al., Chem. Ind. (London) 38, 1597-98 (1966) . Chloromethylated resinε are commercially available from Bio Rad Laboratorieε, Richmond, California and from Lab. Systems, Inc. The preparation of such a resin is described by Stewart et al., "Solid Phase Peptide Synthesiε", supra. BHA and MBHA resin supportε are commercially available and are generally uεed when the deεired polypeptide being εyntheεized haε an unεubεtituted amide at the C-terminuε. Thuε, εolid reεin supports may be any of those known in the art, εuch as one having the formulae: -0-CH ₂ -resin εupport, -NH BHA reεin εupport or -NH-MBHA reεin εupport. When the unεubεtituted amide is deεired, use of a BHA or MBHA resin is preferred, becauεe cleavage directly giveε the amide. In caεe the N-methyl amide iε desired, it can be

generated from an N-methyl BHA resin. Should other substituted amides be desired, the teaching of U.S. Patent No. 4,569,967 can be used, or should still other groups than the free acid be desired at the C-terminus, it may be preferable to synthesize the peptide using classical methods as set forth in the Houben-Weyl text.

The C-terminal amino acid, protected by Boc and by a side-chain protecting group, if appropriate, can_be first coupled to a chloromethylated resin according to the procedure set forth in Chemistry Letters. K. Horiki et al. 165-168 (1978), using KF in DMF at about 60°C. for 24 hours with εtirring, when a peptide having free acid at the C-terminuε is to be synthesized. Following the coupling of the BOC-protected amino acid to the resin support, the α-amino protecting group iε removed, aε by uεing trifluoroacetic acid(TFA) in methylene chloride or TFA alone. The deprotection iε carried out at a temperature between about 0°C and room temperature. Other εtandard cleaving reagents, such as HCl in dioxane, and conditions for removal of specific α-amino protecting groupε may be uεed as described in Schroder & Lubke, "The Peptideε", 1 pp 72-75, Academic Preεε (1965).

After removal of the α-amino protecting group, the remaining α-amino- and side chain-protected amino acids are coupled step-wiεe in the desired order to obtain the intermediate compound defined hereinbefore, or as an alternative to adding each amino acid separately in the synthesis, some of them may be coupled to one another prior to addition to the solid phase reactor. The selection of an appropriate coupling reagent iε within the εkill of the art. Particularly εuitable aε a coupling reagent is N,N'-dicyclohexyl carbodiimide (DCC) .

The activating reagentε uεed in the solid phase synthesis of the peptides are well known in the peptide art. Examples of suitable activating reagents are carbodiimides, εuch as N,N'-diiεopropylcarbodiimide and

N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide. Other activating reagents and their use in peptide coupling are described by Schroder & Lubke supra, in Chapter III and by Kapoor, J. Phar. Sci.. 59, pp 1-27 (1970). Each protected amino acid or amino acid sequence is introduced into the solid phase reactor in about a twofold or more excess, and the coupling may be carried out in a medium of dimethylformamide(DMF) : CH ₂ C1 ₂ (1:1) or in DMF or CH ₂ C1 ₂ alone. In cases where incomplete coupling occurs, the coupling procedure is repeated before removal of the α-amino protecting group prior to the coupling of the next amino acid. The success of the coupling reaction at each stage of the synthesis, if performed manually, is preferably monitored by the ninhydrin reaction, as described by E. Kaiser et al.. Anal. Biochem. 34, 595 (1970). The coupling reactions can be performed automatically, as on a Beckman 990 automatic syntheεizer, using a program such as that reported in Rivier et al. Biopolymers. 1978, 17, pp 1927-1938.

After the desired amino acid sequence has been completed, the intermediate peptide can be removed from the resin support by treatment with a reagent, such as liquid hydrogen fluoride, which not only cleaveε the peptide from the reεin but also cleaves all remaining side chain protecting groups and also the α-amino protecting group at the N-terminus if it was not previouεly removed to obtain the peptide in the form of the free acid. If Met iε preεent in the sequence, the Boc protecting group is preferably first removed using trifluoroacetic acid(TFA) /ethanedithiol prior to cleaving the peptide from the resin with HF to eliminate potential S-alkylation. When using hydrogen fluoride for cleaving, one or more scavengerε, εuch aε aniεole, creεol, dimethyl εulfide, and methylethyl εulfide are included in the reaction veεsel.

Cyclization of the linear peptide iε preferably effected, as opposed to cyclizing the peptide while a part of the peptidoresin, to create bondε between Cyε residues. To effect such a disulfide cyclizing linkage, the fully protected peptide can be cleaved from a hydroxymethylated resin or a chloromethylated resin support by ammonolyεiε, aε iε well known in the art, to yield the fully protected amide intermediate, which iε thereafter suitably cyclized and deprotected; alternatively, deprotection as well as cleavage of the peptide from the above resins or a benzhydrylamine (BHA) resin or a methyl-benzhydrylamine (MBHA) , can take place at 0°C with hydrofluoric acid (HF) , followed by air- oxidation under high dilution conditions.

Thus, in one aspect, the invention also provides a method for manufacturing a synthetic conotoxin peptide of interest by carrying out the following steps: (a) forming a peptide intermediate having the desired amino acid residue sequence and at least one protective group attached to a labile side chain of a residue such as Ser, Thr, Tyr, Aεp, Glu, His, Cyε, Arg or Lyε and optionally having itε C- terminus linked by an anchoring bond to resin support; (b) splitting off the protective group or groups and any anchoring bond from the peptide intermediate to form a linear peptide; (c) creating a cyclizing bond between Cys reεidueε preεent in the linear peptide to create a cyclic peptide; and (d) if deεired, converting the resulting cyclic peptide into a nontoxic salt thereof. Particular side chain protecting groups and resin supports are well known in the art and are disclosed in the earlier-referenced patents.

In order to illuεtrate specific preferred embodiments of the invention in greater detail, the following exemplary work iε provided.

EXAMPLE 1 Conotoxin SEQ ID NO:l (alεo referred to aε J- 020) , having the chemical formula:

H-Gly-Cys-Cyε-Gly-Ser-Tyr-Pro-Aεn-Ala-Ala-Cys-His-Pro- Cys-Ser-Cys-Lys-Asp-Arg-4Hyp-Ser-Tyr-Cys-Gly-Gln-NH ₂ is synthesized by stepwise elongation from the carboxyl terminuε, using the solid phase Merrifield peptide synthesis procedure. Operational details of this general procedure, which are not set forth hereinafter, can be found in Stewart, J.M. and Young, J. , Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, 111., (1984), and in Rivier et al., U.S. Patent No. 5,064,939 (Nov. 12, 1991) [the discloεure of the latter of which iε incorporated herein by reference] . A methylbenzyhydrylamine reεin iε uεed as the solid phase εupport and facilitates production of the amidated peptide. Amino acid residues, in the form of their Boc (tert-butyloxycarbonyl) derivatives, are coupled successively to the resin using dicyclohexylcarbodiimide (DCC) as the coupling or condensing agent. At each cycle of stepwise amino acid addition, the Boc group is removed by acidolysis with 50 percent (v/v) trifluoroacetic acid (TFA) in methylene chloride, using an appropriate εcavenger, εuch aε 1,2 ethanedithiol, thereby expoεing a new α-amino group for the subsequent coupling εtep. More specifically, when an automated machine and about 5 grams of resin are used, following the coupling of each amino acid residue, washing, deblocking and coupling of the next residue are preferably carried out according to the following schedule:

STEP REAGENTS AND OPERATIONS MIX TIMES

MIN.

1 CH ₂ C1 ₂ wash-40 ml . ( 2 times) 3

2 Methanol (MeOH) wash-30 ml . ( 2 timeε) 3

3 CH ₂ C1 ₂ wash-80 ml . ( 3 times) 3

4 50 percent TFA plus 5 percent 1 , 2-ethane- dithiol in CH ₂ Cl ₂ -70 ml . (2 times) 12 5 Iεopropanol wash-40 ml . ( 2 times) 3

6 TEA 12 . 5 percent in CH ₂ Cl ₂ -70 ml .

( 2 times) 5

7 MeOH wash-40 ml. (2 times) 2

8 CH ₂ C1 ₂ waεh-80 ml. (3 timeε) 3 9 Boc-amino acid (10 mmoleε) in 30 ml. of either

DMF or CH ₂ C1 ₂ , depending upon the εolubility of the particular protected amino acid, (1 time) plus DCC (10 mmoles) in CH ₂ C1 ₂ 30-300

Side chain protecting groupε are generally chosen from among the standard set of moderately acid- εtable derivativeε. Such protecting groupε are preferably oneε that are not removed during deblocking by trifluoroacetic acid in methylene chloride; however, all are cleaved efficiently by anhydrous hydrofluoric acid (HF) to releaεe the functional εide chainε. Cysteine residueε in positions 2, 3, 11, 14, 16 and 23 of the peptide are protected by p-methoxy-benzyl (Mob) groups so as to expose sulfhydryls upon deprotection. The phenolic hydroxyl group of Tyr is protected by 2- bromo-benzyloxycarbonyl (Brz) . The side chain of 4- hydroxyproline (4Hyp) is protected by benzyl ether (OBzl) , and it is commercially available in thiε protected form. The εide chain of Arg iε protected with Toε (p-toluenesulfonyl) . The side chain of Asp is protected as the cyclohexyl ester (OChx) , and the

primary amino side chain of Lys is protected with 2- chlorobenzyloxycarbonyl (Clz) . The imidazole nitrogen of His is protected by Tos. Serine is protected by benzyl ether (OBzl) . Asn iε coupled without side chain protection in the presence of hydroxybenzotriazole (HOBt) .

At the end of the synthesis, the following peptide intermediate is obtained: Boc-Gly-Cys(Mob)- Cys(Mob)-Gly-Ser(OBzl)-Tyr(Brz)-Pro-Asn-Ala-Ala- Cys(Mob)-His(Tos)-Pro-Cys(Mob)-Ser(OBzl)-Cys(Mob)- Lys(Clz)-Asp(OChx)-Arg(Tos)-4Hyp(Bzl)-Ser(OBzl)- Tyr(Brz)-Cys(Mob)-Gly-Gln-MBHA resin support. All the side-chain blocking groups are HF-cleavable.

After removing the N-terminal Boc group with

TFA, the linear peptide is cleaved from the resin and deprotected with HF, using 150 milliliters of HF, 16 ml of aniεole and about 4 ml dimethyl sulfide for about 1.5 hours at 0°C, which removes all the remaining protecting groups. Any volatiles are removed by the application of a vacuum, and the peptide is waεhed with ethylether and then diεεolved in 5 percent acetic acid. The solution is then diluted to about 15 liters and pH is adjusted to about 8.0 with diisopropyl ethylamine. It is exposed to air-oxidation in a cold room at about 4°C. for 4 days to form the disulfide cross links or bridges. One drop of mixture is recovered about every 12 hours and added to one drop of a solution containing dithio-bis(2-nitrobenzoic) acid in a molar buffer of K ₂ HP0 ₄ (pH 8) in order to follow the progress of the oxidation reaction (Ellman test) . During the whole reaction, the pH was maintained at 8 by addition of diisopropylethylamine. After 50 hours, the absence of yellow coloration is obεerved in the test with dithio- bis(2-nitrobenzoic) acid.

After formation of the disulfide bridges, the cyclized pool of peptides is applied to a Bio-Rex-70 column (5 x 15 cm) , waεhed in distilled water (100 ml) , and eluted with 50% acetic acid. The cyclized peptide fractions are collected and lyophilized.

The lyophilized peptide fractionε are then purified by preparative or εemi-preparative HPLC aε deεcribed in Rivier, et al., J. Chromatography. 288, 303-328 (1984); and Hoeger, et al., BioChromatography, 2., 3, 134-142 (1987). The chromatographic fractions are carefully monitored by HPLC, and only the fractions showing substantial purity are pooled.

The peptide is judged to be homogeneouε by reverεed-phase high performance liquid chromatography using a Waters HPLC system with a 0.46 x 25 cm. column packed with 5μm C ₁₈ silica, 30θA pore size. The determination iε run at room temperature using gradient conditions with 2 bufferε. Buffer A iε an aqueouε trifluoroacetic acid (TFA) εolution conεiεting of 1.0 ml. of TFA per 1000 ml. of εolution. Buffer B iε 1 ml TFA diluted to 400 ml with H ₂ 0 which is added to 600 ml. of acetonitrile. The analytical HPLC was run under gradient conditions which vary uniformly from 20 volume percent (v/o) Buffer B to 35 v/o Buffer B over 10 minutes, at a constant flow rate of 2 ml. per minute; the retention time for the biologically active cyclic conotoxin is 10.6 minuteε.

The product is also characterized by amino acid analysiε and by toxicity teεtε. One microgram of the synthetic toxin injected intracerebrally (IC) in a mouse iε lethal in leεε than 10 minuteε εhowing that the synthetic product is highly toxic, and thus εynthesis by the deεcribed method, if followed by air-oxidation, achieveε the correct diεulfide pairing arrangement to aεεure biological activity. The εynthetic peptide is shown to be substantially identical with the native

conotoxin as a result of coelution on HPLC, amino acid analysis and biological activity. This peptide binds to and inhibits the function of the acetylcholine receptor, thereby causing paralyεiε and thereafter death. It can be uεed in aεεayε for the acetylcholine receptor.

EXAMPLE 2 Conotoxin SEQ ID NO:2 (alεo referred to as J-005) , having the chemical formula: H-pGlu-Lys-Ser-Leu-Val-Pro-Ser-Val-Ile-Thr-Thr-Cys-Cys- Gly-Tyr-Asp-4Hyp-Gly-Thr-Met-Cys-4Hyp-4Hyp-Cys-Arg-Cys- Thr-Aεn-Ser-Cyε-NH ₂ iε synthesized by stepwiεe elongation from the carboxyl terminuε, using the solid phase synthesis procedure as set forth in Example 1 and the same methyl benzyhydrylamine resin.

The side chains of hydroxyproline, threonine and serine are protected by benzyl ether (Bzl) .

At the end of the synthesis, the following peptide intermediate is obtained: Boc-pGlu-Lys(Clz)- Ser(Bzl)-Leu-Val-Pro-Ser(Bzl)-Val-Ile-Thr(Bzl)-Thr(Bzl)- Cys(Mob)-Cys(Mob)-Gly-Tyr(Brz)-Asp(OChx)-4Hyp(Bzl)-Gly- Thr(Bzl)-Met-Cys(Mob)-4Hyp(Bzl)-4Hyp(Bzl)-Cys(Mob)- Arg(Tos)-Cys(Mob)-Thr(Bzl)-Asn-Ser(Bzl)-Cys(Mob)-MBHA resin support. All the side-chain blocking groups are HF-cleavable.

After removing the N-terminal Boc group with TFA, the linear peptide is cleaved from 3 grams of the reεin and deprotected, uεing 100 milliliterε of HF, 1 ml of aniεole and about 4 ml dimethyl sulfide for about 1.5 hours at 0°C, which removes all the remaining protecting groups. Any volatiles are removed by the application of a vacuum, and the peptide is washed with ethylether and then extracted with 10 percent acetic acid containing 10% cyanomethane. The solution is then diluted to about 4 liters and a pH of about 6.95. The solution is exposed to air-oxidation in a cold room at

about 4°C. for a time sufficient to completely oxidize by forming diεulfide croεslinks or bridges, i.e., a period of about 1 to 2 weeks.

After formation of the disulfide bridges, the cyclized pool of peptides is applied to a Bio-Rex-70 column (5 x 15 cm) and eluted with 50% acetic acid. The cyclized peptide fractions are collected and lyophilized. The synthetic peptide is shown to be substantially identical with the native conotoxin as a result of coelution on HPLC, amino acid analysis and biological activity, which comparison is made with the native conotoxin following deglycosylation to remove the carbohydrate linked to Ser in the 7-position which increaεeε bioactivity. When injected IC into mice, the peptide causes mice to become εpaεtic and to εuffer paralyεis. It is thus known to have high affinity and εpecificity for a particular receptor and can be used to target this receptor and in assayε for this receptor.

EXAMPLE 3 The peptide OB-34 (SEQ ID NO:3) is produced by using the synthesis as generally set forth in Example 1. The peptide in question haε the following formula: H-Cys-Cys-Gly-Val-4Hyp-Aεn-Ala-Ala-Cyε-Pro-4Hyp-Cyε-Val- Cyε-Asn-Lys-Thr-Cyε-Gly-NH ₂

The εyntheεiε is carried out on an MBHA resin, and Boc is used to protect the α-amino groups. The same side chain protecting units are used as described hereinbefore.

About 4-1/2 grams of the peptide-resin iε treated with 5 milliliterε of aniεole, 1 milliliter of methylethyl εulfide, and 60 milliliterε of HF for 1/2 hour at -20°C and 1 hour at 0°C. The peptide is then extracted and dissolved in 4.5 liters of ammonium acetate buffer, a solution containing about 10 grams of

ammonium acetate at a pH of about 4.3. pH iε adjuεted to about 7.75 with ammonium hydroxide, and the εolution is maintained in a cold room at about 4°C. for a sufficient length of time to allow complete air- oxidation to occur. Purification is then carried out as previously deεcribed with reεpect to Example 2, and the purified peptide iε εubjected to analytical HPLC. It iε found to exhibit a single peak with both a gradient flow and with isocratic flow of appropriate buffers. The purity of the compound was estimated to be greater than about 99 percent. The εynthetic peptide coelutes with the native peptide on HPLC.

Injection of 1 microgram of the synthetic peptide OB-34 intracerebrally into a mouse shows that the mouse exhibits a reproducible physical effect indicative of binding to a specific receptor and confirms that the air-oxidation produces appropriate crosε-linking so that the synthetic conotoxin exhibits biological potency. It is thus known to have high affinity and specificity for a particular receptor and can be used to target this receptor and in asεays for this receptor.

EXAMPLE 4 The peptide J-019 (SEQ ID NO:4) is syntheεized uεing the procedure aε deεcribed with reεpect to Example 1. The synthetic peptide has the following formula: H-Gly-Cys-Cys-Gly-Ser-Tyr-4Hyp-Asn-Ala-Ala-Cyε-Hiε-4Hyp- Cyε-Ser-Cyε-Lys-Asp-Arg-4Hyp-Ser-Tyr-Cyε-Gly-Gln-NH ₂ An MBHA reεin is used, and Boc is uεed to protect the α-amino groupε of each of the amino acidε employed in the εyntheεis. Side chain protecting groups as set forth with respect to Example 1 are similarly employed. Cleavage from the resin and air-oxidation to

carry out cyclicization are performed as εet forth in Example 1.

The cyclic peptide is purified using the procedure set forth in Example 1 and checked for purity via analytical HPLC, which shows that a subεtantially pure εynthetic material iε obtained. The synthetic peptide is shown to be substantially identical with the native conotoxin as a result of coelution on HPLC, amino acid analysis and biological activity. Injection of the peptide intracerebrally into a mouse shows an initial attack of violent scratching followed by paralysis and ultimate death, confirming that air-oxidation can produce appropriate cross-linking so that the εynthetic conotoxin exhibits biological potency. It is thus known to have high affinity and specificity for a particular receptor and can be used to target this receptor and in asεayε for thiε receptor.

EXAMPLE 5 The procedure of Example 4 iε repeated with a single change of the amino acid in the 13-position to εubstitute proline for 4-hydroxyproline and thereby syntheεize the peptide J-026 (SEQ ID NO:5). The εynthetic peptide haε the following formula: H-Gly-Cyε-Cys-Gly-Ser-Tyr-4Hyp-Asn-Ala-Ala-Cyε-Hiε-Pro- Cyε-Ser-Cyε-Lyε-Aεp-Arg-4Hyp-Ser-Tyr-Cyε-Gly-Gln-NH ₂

Cleavage from the reεin and air-oxidation to carry out cyclicization are performed aε εet forth in Example 1. Analytical HPLC εhowε the substantially pure compound is obtained. The synthetic peptide is shown to be substantially identical with the native conotoxin as a result of coelution on HPLC, amino acid analysiε and biological activity. Teεting by IC injection into a mouse results in violent movements followed by paralyεis and death. It iε thuε known to have high affinity and

specificity for a particular receptor and can be used to target this receptor and in asεays for this receptor.

EXAMPLE 6 The syntheεiε of peptide OB-26 (SEQ ID NO:6) is carried out using a procedure generally the same as that described with reεpect to Examples 1 and 3. The synthetic peptide has the following formula: H-Cys-Cys-Gly-Val-4Hyp-Asn-Ala-Ala-Cyε-Hiε-4Hyp-Cyε-Val- Cys-Lys-Asn-Thr-Cys-NH ₂

Cleavage from the MBHA reεin and air-oxidation are carried out aε set forth in Example 1. HPLC purification of the crosε-linked peptide is carried out in a similar manner. The resultant εynthetic peptide is checked by analytical HPLC and shown to constitute a substantially pure compound. The synthetic peptide is shown to be subεtantially identical with the native conotoxin aε a reεult of coelution on HPLC, amino acid analysis and biological activity. Injection of 1 microgram of the synthetic peptide IC into a mouse resultε in a reproducible physical effect, which verifies that the appropriate disulfide linkages are achieved during the air-oxidation step. It is believed that the peptide haε high affinity and εpecificity for a particular receptor and that it can be uεed to target this receptor and to assay for this receptor.

EXAMPLE 7 Synthesis of the peptide J-029 (SEQ ID NO:7) is carried out on a chloromethylated resin in the same general manner as set forth in Example 6. The synthetic peptide has the following formula:

H-Gly-4Hyp-Ser-Phe-Cys-Lyε-Ala-Aεp-Glu-Lys-4Hyp-Cys-Glu - Tyr-His-Ala-Aεp-Cyε-Cyε-Aεn-Cyε-Cys-Leu-Ser-Gly-Ile-Cys - Ala-Hyp-Ser-Thr-Asn-Trp-Ile-Leu-Pro-Gly-Cyε-Ser-Thr-Ser- Ser-Phe-Phe-Lyε-Ile-OH

The peptide is cleaved from the resin with anisole, methylethyl sulfide and HF, and air-oxidation is then carried out under the conditions as generally set forth in Example 1 in order to obtain the cyclic compound. Thereafter, purification is carried out using HPLC as set forth hereinbefore. Ultimate subjection of the purified peptide to analytical HPLC shows that a subεtantially pure compound is obtained. The synthetic peptide iε εhown to be substantially identical with the native conotoxin as a result of coelution on HPLC, amino acid analysiε and biological activity.

Injection of a dose of about 1 microgram of the synthetic conotoxin IC into a mouse shows substantially immediate paralysiε occurring. It iε thuε known to have high affinity and εpecificity for a particular receptor and can be uεed to target this receptor and in asεayε for thiε receptor.

EXAMPLE 8 The εyntheεiε of peptide OB-20 (SEQ ID NO:8) having the formula:

H-Gly-Cys-Cyε-Ser-Hiε-Pro-Ala-Cys-Ser-Gly-Lys-Tyr-Gln- Gla-Tyr-Cyε-Arg-Gla-Ser-NH ₂ iε carried out generally aε set forth in Example 3 using an Fmoc strategy on a 2,4dimethoxy-alkoxybenzyl a ine reεin.

The peptide iε cleaved from the reεin uεing a mixture of TFA, thioaniεole, water and DCM in the following volume ratios: 40:10:1:44. Cleavage is carried out for about 8 hours at 37°C. Following cleavage, air-oxidation is carried out to cyclize the peptide as set forth in Example 1.

Purification of the cyclized peptide is carried out as set forth hereinbefore. Subjection of the purified peptide to HPLC and amino acid analysis showε that a peptide having a purity of greater than 95 percent is obtained, which has the expected ratio of

reεidueε when εubjected to amino acid analysis. The synthetic peptide coelutes with the native peptide on HPLC.

Injection of 1 microgram of the εynthetic peptide OB-20 intracerebrally into a mouse showε that the mouεe exhibitε a reproducible phyεical effect and confirmε that air-oxidation produceε appropriate cross- linking so that the synthetic conotoxin exhibits biological potency. It is thus known to have high affinity and specificity for a particular receptor and can be used to target this receptor and in assayε for thiε receptor.

EXAMPLE 9 A εyntheεiε, aε generally set forth in Example

1, is carried out using about 25 grams of a chloromethylated polystyrene resin of the type generally commercially available to produce peptide J-021 (SEQ ID NO:9) which haε the following formula: H-Hiε-4Hyp-4Hyp-Cyε-Cys-Leu-Tyr-Gly-Lys-Cys-Arg-Arg-Tyr- 4Hyp-Gly-Cys-Ser-Ser-Ala-Ser-Cyε-Cys-Gln-OH.

Similar side chain protecting groups are provided as described in Example 1, and the hydroxyl side chain of 4-hydroxyproline is protected as the benzyl ether. Coupling of the N-terminal His reεidue iε carried out uεing Boc-Hiε(Toε) diεεolved in DMF and uεing about 3 millimoles of benzotriazol-1-yl-oxy- triε(dimethylamino)phoεphonium hexafluorophoεphate (BOP) aε a coupling agent. After the final His residue is coupled to the peptide-resin, the Boc group iε removed uεing 45 percent TFA in methylene chloride. The peptide-reεin iε then treated with anisole and methylethyl sulfide and HF. Five grams of resin are treated with 10 milliliters of anisole, one ml of methylethyl εulfide and 125 ml of HF for 1/2 hour at -20°C and 1 hour at 0°C. The cleaved

peptide iε then extracted uεing 200 milliliterε of 50 percent acetic acid at a temperature below 0°C. Thereafter, the extracted peptide iε dissolved in 8 literε of 1 percent ammonium acetate at a pH of about 4.35. The pH iε raiεed to about 7.74 with ammonium hydroxide, and air-oxidation iε effected aε deεcribed in Example 1.

Purification is carried out as described in Example 1, and then purity is checked using analytical HPLC. The peptide is applied to a reversed phase C ₁₈ column, and then eluted by subjecting the column to a gradient of buffers A and B at a flow rate of about 0.21 milliliters per minute, which gradient changes uniformly from 0 percent buffer B to 20 percent buffer B over a time period of 20 minuteε. Buffer A iε a 1 percent aqueous solution of TFA, and buffer B is 0.1% TFA and 70% acetonitrile. This HPLC εhowε that the peptide eluteε at about 18.6 minutes and has a purity of greater than 99 percent. The synthetic peptide coelutes with the native peptide on HPLC. Amino acid analysis of the pure peptide showε that the expected reεidueε are obtained. It iε believed that testing will show this peptide to have high affinity and specificity for a particular receptor so that it can be used to target this receptor or to assay for this receptor.

EXAMPLE 10 The peptide J-010 (SEQ ID NO:10) is synthesized using the procedure as generally set forth with respect to Example 8 uεing an Fmoc protection εtrategy. The synthetic peptide has the following formula: H-Cys-Lys-Thr-Tyr-Ser-Lys-Tyr-Cys-Gla-Ala-Asp-Ser-Gla- Cys-Cyε-Thr-Gla-Gln-Cyε-Val-Arg-Ser-Tyr-Cys-Thr-Leu-Phe- NH ₂ . The peptide is cleaved from the resin uεing a mixture of TFA, thioaniεole, water and DCM in the

following volume ratios: 40:10:1:44. Cleavage is carried out for about 8 hours at 37°C. Following cleavage, air-oxidation is carried out to cyclize the peptide as previously described. Purification of the cyclized peptide iε carried out aε set forth hereinbefore, and subjection of the purified peptide to HPLC εhowε that a substantially pure peptide is obtained. The εynthetic peptide iε εhown to be substantially identical with the native conotoxin as a result of coelution on HPLC, amino acid analysiε and biological activity. Injection of about 1 microgram of the εynthetic peptide intracerebrally into a mouse shows that the mouse begins rapid running and stretching, ultimately resulting in death. It is thus known to have high affinity and εpecificity for a particular receptor and can be uεed to target thiε receptor and in assayε for this receptor.

EXAMPLE 11 A syntheεis as generally performed in Example 1 is carried out to produce peptide J-008 (SEQ ID NO:11) having the formula:

H-Ser-Thr-Ser-Cys-Met-Glu-Ala-Gly-Ser-Tyr-Cys-Gly-Ser- Thr-Thr-Arg-Ile-Cys-Cys-Gly-Tyr-Cys-Ala-Tyr-Phe-Gly-Lys- Lys-Cyε-Ile-Asp-Tyr-Pro-Ser-Asn-OH.

The C-terminal reεidue in the peptide is Asn in its free acid form. An MBHA reεin waε used along with the incorporation of Boc-protected Asp through its β- carboxylic group. Cleavage from the resin and cyclization iε carried out aε in Example 1. The final product is similarly purified to homogeneity by HPLC, and amino acid analysis of the purified peptide gives the expected resultε. The synthetic peptide coelutes with the native peptide on HPLC.

The synthetic toxin iε injected IC into a mouse, and it proves lethal in less than 10 minutes, confirming that the synthetic product is highly toxic and that the stated synthesis produces a compound having biological activity. It is thus known to have high affinity and specificity for a particular receptor and can be used to target thiε receptor and in assayε for thiε receptor.

EXAMPLE 12

Syntheεiε of conotoxin SEQ ID NO:12 (also referred to as J-017) , having the formula: H-Gly-Glu- Gla-Gla-Val-Ala-Lyε-Met-Ala-Ala-Gla-Leu-Ala-Arg-Gla-Aεn- Ile-Ala-Lyε-Gly-Cyε-Lyε-Val-Aεn-Cyε-Tyr-Pro-OH iε carried out generally εimilarly to that of Example 1 but uεing the modificationε deεcribed hereinafter.

A commercially available p-alkoxybenzyl alcohol resin is used for the syntheεiε, which is a standard resin used in solid phaεe εyntheses employing the Fmoc- amino acid εtrategy. Fluorenylmethyloxycarbonyl (Fmoc) iε uεed to protect the α-amino groupε of each of the amino acidε, and Boc protection is used for the side- chain amino groups of Lys. The Tyr side chain is protected by O-tBu, and the Cys side chain is protected by diphenylmethyl (trityl) . The carboxyl side chain of Glu and the side chainε of Gla are protected by O-t-Bu aε deεcribed hereinafter. Arg iε protected by 4- methoxy-2,3,6-trimethylbenzeneεulfonyl (Mtr) .

Fmoc-L-Gla(0-t-Bu) ₂ -OH iε prepared aε εet forth hereinafter. Condenεation of Z-L-Ser(Tos)-OCH ₃ with di-tert-butyl malonate, to give Z-DL-Gla(0-t-Bu) ₂ -OCH ₃ , is carried out by a modification of the procedure of Rivier et al. Biochemiεtry 26, 8508-8512 (1987) . Sodium hydride is rinsed twice with pentane, suspended in absolute benzene, and then added to the benzene solution of di-tert-butyl malonate. The reaction is allowed to

proceed to completion with 10 minutes of reflux. The resulting suspension is cooled in an ice bath, and the Z-L-Ser(Toε)-0CH ₃ diεsolved in benzene/tetrahydrofuran is added under an argon atmosphere with vigorouε stirring and continued cooling at 0°C. for 2 hours. Stirring is maintained for additional 48 hours at room temperature. At this time, the suspension is cooled and washed successively with ice water, 1 N HCl, and water. After rotary evaporation at room temperature, the oil is disεolved in benzene, and pentane iε added to initiate cryεtallization. The yield iε 40-60% for a preparation of 0.5 mole. The methyl eεter iε hydrolyzed by dissolving in alcohol and adding 1.2 equiv of KOH disεolved in water/ethanol. The εolution is allowed to remain at room temperature for several days; the reaction is monitored by HPLC using a C ₁₈ 5-μm column, with 0.1% TFA-acetonitrile as the solvent. When the reaction is complete, the solution is evaporated at room temperature, and the product extracted with ethyl acetate after the addition of NaHS0 ₄ . The ethyl acetate extract is dried over Na ₂ S0 ₄ and evaporated under reduced preεεure; the yield iε 80-90%.

The D- and L-iεomerε are reεolved by crystallization of the quinine salt of the D-isomer. Z-DL-di-t-Bu-Gla-OH in ethyl acetate is reacted with an equivalent amount of quinine. The crystalε are εeparated from the mother liquid, and the Z-D-di-t-Bu-Gla-OH iε recryεtallized from ethyl acetate. The quinine εalt iε εuspended in ether, and quinine is removed by the addition of a 20% citric acid solution at 0°C. The same procesε is used to remove quinine from the liquid phaεe. The L-iεomer is precipitated in the form of itε ephedrine εalt from ethyl acetate-pentane and recrystallized (Marki et al., Helv. Chim. Acta. 60, 798-800, 1977) . Elimination of ephedrine by acid extraction, hydrogenation of the Z group, and

introduction of the Fmoc are all standard laboratory procedures. Optical purity of the L- and D-isomers of Fmoc-Gla(0-t-Bu) ₂ -OH is assessed after hydrolysis to Glu (6 N HC1, 110°C, 20 hours) , and each iε approximately 99% pure.

The coupling of the Fmoc-protected amino acidε to the resin is accomplished using a εchedule generally εimilar to that εet forth in Example 1 but removing the Fmoc group via the use of a 20 percent solution (v/v) of freshly distilled piperidine in dimethylformamide (DMF) for 10 minutes. Thorough resin waεhing iε accompliεhed by repeated application of DMF, methanol, or dichloromethane (DCM) . Couplingε are mediated by DCC in either DCM, DMF, or mixtures thereof, depending upon the solubility of the particular amino acid derivative. Fmoc-Asn is incorporated into the peptide with an unprotected εide chain, in the preεence of 2 equiv of HOBT, and iε coupled in DMSO/DMF or DMSO/DCM.

The peptide iε releaεed from 4 gramε of the peptide reεin aε the C-terminal free acid by treatment with a freεhly prepared mixture of TFA, thioaniεole, H ₂ 0, EDT and DCM (40/18/1/2/49) (40 ml) at about 37°C for 6-8 hourε. Trial cleavages on small amountε demonstrate that the peptide is freed and that all side-chain protection, including the difficult Mtr group, are removed while Gla remains intact.

The peptide is precipitated from the cleavage solution after extraction with methyl tert-butyl ether. The peptide is then disεolved in diεtilled water, the pH of the reεulting solution is adjusted to approximately

7-8 with dilute ammonium hydroxide, after separating the reεin by filtration. Formation of the diεulfide cross¬ link is carried out on the crude peptide product using a liquid phase, air-oxidation step in a cold room as described with respect to Example 1. The crude peptide is purified by preparative HPLC using a preparative

cartridge (15-20 μm, 300 A Vydac C ₁₈ ) and a TEAP buffer, pH 2.25, and also with a 0.1% TFA buffer using appropriate gradientε of acetonitrile. Highly purified fractionε are pooled and lyophilized, yielding peptide aε itε TFA εalt. Optical rotation in 1% acetic acid meaεures [α] _D = -64° (c = 1) at 20°C. Amino acid analysis gives the expected values. FAB masε spectrometry iε performed on the peptide, and the spectrum shows a protonated molecular ion (MH ⁺ ) at m/z = 3097.4 corresponding to the calculated monoisotopic peptide of 3097.36. A chromatogram of the crude preparation after TFA cleavage and deprotection illustrateε that the major product iε particularly pure and that only a relatively εmall amount of hydrophobic impuritieε are preεent. Sequence analyεiε giveε the expected reεidue at each cycle, except for blankε with Gla residues, confirming that the pure target peptide is obtained. The synthetic peptide coeluteε with the native peptide on HPLC. When injected IC into young mice, it causes sleeping; however, when injected into older mice, it causeε hyperactivity. It iε thuε known to have high affinity and εpecificity for a particular receptor and can be used to target this receptor and in assays for this receptor, tentatively identified as the NMDA receptor. It can be used to provide neuroprotection.

EXAMPLE 13 A synthesis of the linear peptide J-004 (SEQ ID NO:13) is carried out on an MBHA resin using the procedure as generally set forth in Example 1. The linear peptide J-004 haε the following formula: H-Glu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lyε-Lyε-Pro- Tyr-Ile-Leu-NH ₂ . The ultimate linear peptide is purified and subjected to amino acid analysiε; it εhows that the

expected residues are obtained in the peptide sequence. The synthetic peptide coelutes with the native peptide on HPLC, after the native conotoxin has been deglycosylated to remove the carbohydrate which is linked to Thr in the 10-position which appearε to increaεe bioactivity. Teεting of the εynthetic peptide by injection IC into a mouse εhowε that the mouse quickly becomeε sluggish and unable to stand or function normally, which demonstrateε that the synthetic peptide has the expected biological potency. It is thus known to have high affinity and specificity for a particular receptor and can be used to target this receptor and in aεεayε for thiε receptor.

Theεe εynthetic peptideε, for ad iniεtration to humanε, εhould have a purity of at leaεt about 95 percent (herein referred to a εubεtantially pure) , and preferably have a purity of at leaεt about 98 percent. Purity for purposes of this application refers to the weight of the intended peptide as compared to the weight of all peptide fragments present. These synthetic peptideε, either in the free form or in the form of a nontoxic salt, are commonly combined with a pharmaceutically or veterinarily acceptable carrier to create a composition for administration to animals, including humanε, or for uεe in in vitro aεεayε. In vivo adminiεtration εhould be carried out by a phyεician and the required doεage will vary with the particular objective being purεued. In thiε reεpect, guidelineε have been developed for the uεe of other conotoxinε εuch aε conotoxin GI and εuch are well known in thiε art are employed for the particular purpose of use.

As indicated hereinbefore, DNA encoding the amino acid structure of any of these conotoxins can be used to produce the proteins recombinantly as well as to afford different varieties of plants with pesticidal propertieε.

To syntheεize a protein having the desired conotoxin amino acid reεidue εequence by recombinant DNA, a double-εtranded DNA chain which encodeε the sequence might be εynthetically constructed. Although it is nowadays felt that PCR techniques would be method of choice to produce DNA chains, a DNA chain encoding the deεired εequence could be designed using certain particular codonε that are more efficient for polypeptide expression in a certain type of organism, i.e. selection might employ those codons which are most efficient for expression in the type of organism which is to serve as the host for the recombinant vector. However, any correct set of codons will encode a deεired product, although perhapε εlightly less efficiently. Codon selection may also depend upon vector construction considerationε; for example, it may be neceεεary to avoid placing a particular restriction site in the DNA chain if, subsequent to inserting the synthetic DNA chain, the vector is to be manipulated using the restriction enzyme that cleaves at such a site. Also, one should of course avoid placing restriction siteε in the DNA chain if the host organism, which is to be transformed with the recombinant vector containing the DNA chain, is known to produce a restriction enzyme that would cleave at such a site within the DNA chain.

To assemble such a synthetic, nonchromosomal, conotoxin-encoding DNA chain, oligonucleotideε are conεtructed by conventional procedureε such as those described in J. Sambrook et al., Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Presε, New York (1989) (hereinafter, Sambrook et al.). Senεe and antisense oligonucleotide chains, up to about 70 nucleotide residueε long, are synthesized, preferably on automated synthesizers, such as the Applied Biosyεtem Inc. Model 380A DNA εyntheεizer. The oligonucleotide chainε are conεtructed εo that portionε of the sense and

antiεenεe oligonucleotides overlap, associating with each other through hydrogen bonding between complementary base pairs and thereby forming double stranded chains, in most caseε with gapε in the strands. Subsequently, the gaps in the strands are filled in, and oligonucleotides of each εtrand are joined end to end with nucleotide triphosphates in the presence of appropriate DNA polymerases and/or with ligaseε.

Aε an alternative to εuch stepwise construction of a synthetic DNA chain, the cDNA corresponding to the desired conotoxin may be cloned. Aε iε well known, a cDNA library or an expreεεion library is produced in a conventional manner by reverse tranεcription from meεεenger RNA (mRNA) from suitable tisεue from the cone snail of interest. To select clones containing desired sequences, a hybridization probe or a mixed set of probes which accommodate the degeneracy of the genetic code and correεpond to a εelected portion of the protein of intereεt are produced and uεed to identify clones containing such sequences. Screening of such an expreεεion library with antibodieε made against the protein may also be used, either alone or in conjunction with hybridization probing, to identify or confirm the preεence of DNA εequenceε in cDNA library clones which are expressing the protein of interest. Such techniques are taught, for example in Sambrook et al., supra.

In addition to the protein-encoding sequenceε, a DNA chain εhould contain additional εequences depending upon vector construction considerationε. Typically, a εyntheεized DNA chain has linkers at its ends to facilitate insertion into restriction sites within a cloning vector. A DNA chain may be constructed so as to encode the protein amino acid sequences as a portion of a fusion polypeptide; and if εo, it will generally contain terminal εequences that encode amino acid reεidue εequenceε that εerve as proteolytic

proceεεing εiteε, whereby the deεired polypeptide may be proteolytically cleaved from the remainder of the fusion polypeptide. The terminal portions of the synthetic DNA chain may also contain appropriate start and stop signals.

Accordingly, a double-stranded conotoxin-encoding DNA chain is constructed or modified with appropriate linkers for its insertion into a particular appropriate cloning vector. The cloning vector that is to be recombined to incorporate the DNA chain is selected appropriate to its viability and expresεion in a host organism or cell line, and the manner of insertion of the DNA chain depends upon factors particular to the host. For example, if the DNA chain is to be inserted into a vector for insertion into a prokaryotic cell, such aε E. coli, the DNA chain will be inserted 3' of a promoter sequence, a Shine-Delgarno sequence (or ribosome binding site) that is within a 5' non-tranεlated portion and an ATG start codon. The ATG start codon is appropriately spaced from the

Shine-Delgarno sequence, and the encoding sequence is placed in correct reading frame with the ATG start codon. The cloning vector also provides a 3' non-translated region and a translation termination site. For insertion into a eukaryotic cell, such as a yeast cell or a cell line obtained from a higher animal, the conotoxin-encoding oligonucleotide sequence is appropriately spaced from a capping site and in correct reading frame with an ATG start signal. The cloning vector also provideε a 3' non-tranεlated region and a translation termination site.

Prokaryotic transformation vectors, such aε pBR322, pMB9, Col El, pCRl, RP4 and lambda-phage, are available for inεerting a DNA chain of the length which encodeε conotoxin with εubεtantial aεεurance of at leaεt some expresεion of the encoded polypeptide. Typically,

εuch vectorε are conεtructed or modified to have one or more unique restriction siteε appropriately poεitioned relative to a promoter, εuch aε the lac promoter. The DNA chain may be inserted with appropriate linkers into such a restriction εite, with εubεtantial aεεurance of production of desired protein in a prokaryotic cell line transformed with the recombinant vector. To assure proper reading frame, linkers of various lengthε may be provided at the endε of the protein-encoding εequences. Alternatively, cassettes, which include sequences, such as the 5' region of the lac Z gene (including the operator, promoter, transcription εtart site, Shine- Delgarno sequence and translation initiation εignal) , the regulatory region from the tryptophane gene (trp operator, promoter, ribosome binding site and translation initiator) , and a fusion gene containing these two promoters called the trp-lac or commonly called the Tac promoter are available into which the synthetic DNA chain may be conveniently inserted and then the casεette inserted into a cloning vector of choice.

Similarly, eukaryotic transformation vectors, εuch aε, the cloned bovine papillo a viruε genome, the cloned genomeε of the murine retroviruεes, and eukaryotic caεεettes, such as the pSV-2 gpt system

(described by Mulligan and Berg, Nature 277, 108-114, 1979) , the Okayama-Berg cloning system (Mol. Cell Biol. 2 _./ 161-170, 1982) , and the expression cloning vector recently described by Genetics Institute (Science 228 , 810-815, 1985) , are available which provide subεtantial assurance of at leaεt εome expreεεion of conotoxin in the tranεformed eukaryotic cell line.

Aε previously mentioned, a convenient way to ensure production of a protein of the length of the conotoxins of interest is to produce the protein initially as a segment of a gene-encoded fuεion protein.

In such case, the DNA chain is constructed so that the expresεed protein haε enzymatic proceεεing εiteε flanking the conotoxin amino acid reεidue εequenceε. A conotoxin-encoding DNA chain may be inserted, for example, into the beta-galactosidase gene for insertion into E. coli. in which case, the expresεed fuεion protein is subεequently cleaved with proteolytic enzymes to release the conotoxin from beta-galactosidase peptide εequenceε. An advantage of inserting the protein-encoding εequence εo that the desired sequence is expressed as a cleavable segment of a fusion protein, e.g. aε the conotoxin εequence fused within the beta-galactosidaεe peptide εequence, iε that the endogenouε protein into which the desired conotoxin sequence is inserted is generally rendered non-functional, thereby facilitating selection for vectors encoding the fusion protein.

The conotoxin proteinε may alεo be reproduced in yeast using known recombinant DNA techniques. For example, a suitable plasmid, amplified in an E. coli clone, is isolated and cleaved with Eco Rl and Sal I. Thiε digeεted plasmid is electrophoresed on an agaroεe gel allowing for the separation and recovery of the amplified insert of interest. The insert is inserted into the plasmic pYEp, a εhuttle vector which can be uεed to transform both E. coli and Saccharomvces cerevisiae yeast. Insertion of the synthetic DNA chain at this point asεures that the DNA sequence is under the control of a promoter, in proper reading frame from an ATG signal and properly spaced relative to a cap site. The shuttle vector is used to tranεform URA3, a strain of S_i. cerevisiae yeast from which the oratate monophosphate decarboxylase gene iε deleted.

The transformed yeast is grown in medium to attain log growth. The yeast is separated from its culture medium, and cell lysates are prepared. Pooled

cell lysates are determined by RIA to be reactive with antibody raised against the conotoxin, demonstrating that a protein containing protein segment is expressed within the yeast cells.

The production of conotoxins can be carried out in both prokaryotic and eukaryotic cell lines to provide protein for biological and therapeutic use. While conotoxin syntheεis is eaεily demonεtrated uεing either bacteria or yeast cell lines, the synthetic genes should be insertable for expresεion in cellε of higher animals, such as mammalian tumor cellε, and in plants. Such mammalian cells may be grown, for example, aε peritoneal tumorε in host animals, and certain conotoxins may be harvested from the peritoneal fluid. The cloned DNA is insertable into plant varieties of intereεt where the plant utilizeε it aε a plant defense gene, i.e. it produces sufficient amounts of the pesticide of interest to ward off insectε or the like that are natural predators to such plant εpecies.

Although the above examples demonεtrate that conotoxinε can be synthesized through recombinant DNA techniques, the examples do not purport to have maximized conotoxin production. It is expected that subεequent εelection of more efficient cloning vectorε and hoεt cell lineε will increaεe the yield, and known gene amplification techniqueε for both eukaryotic and prokaryotic cellε may be used to increase production. Secretion of the gene-encoded protein from the host cell line into the culture medium is also considered to be an important factor in obtaining certain of the εynthetic proteinε in large quantitieε.

Although the invention has been described with regard to certain preferred embodiments, it should be understood that various changes and modifications as

would be obvious to one having the ordinary skill in the art may be made without departing from the scope of the invention which is set forth in appended claims. For example, substitution of various of the amino acid residues depicted in the amino acid sequenceε by reεidueε known to be equivalent with those residues can be effected to produce equivalent peptides having εimilar biological activitieε. Moreover, it is known that additional substitutions in the amino acid sequence generally throughout the C-terminal portion of the peptide, i.e. within about 1/3 of the length of the conotoxin nearest its C-terminus, can be effected in order to produce conotoxins having phylogenetic specificity; thus, such substitutions in this region can be carried out to produce valuable equivalent structures. The C-terminus of many of the illustrated peptideε iε amidated, and the incluεion of a substituted amide at the C-terminus of such peptides, as described hereinbefore, is considered to create an equivalent conotoxin.

Particular features of the invention are emphasized in the claims which follow.

SEQUENCE LISTING

(1) GENERAL INFORMATION: (i) APPLICANT:

NAME: OLIVERA, Baldomero M. STREET: 1370 Bryan Ave. CITY: Salt Lake City STATE: Utah

COUNTRY: United States POSTAL CODE (ZIP): 84105

NAME: RIVIER, Jean E.F. STREET: 9674 Blackgold Rd. CITY: La Jolla STATE: California COUNTRY: United States POSTAL CODE (ZIP): 92037

NAME: CRUZ, Lourdes J. #403

s 103

NAME: ABOGADIE, Fe

STREET: 2225 Ridge Ave., #2N

CITY: Evanston

STATE: Illinois

COUNTRY: United States

POSTAL CODE (ZIP): 60201 E. So.

es 4102

NAME: DYKERT, John STREET: 704 Barsby Street CITY: Vista STATE: California COUNTRY: United states POSTAL CODE (ZIP): 92084

NAME: TORRES, Josep L.

STREET: Pssg. Maragall 296, 2-1

CITY: 08031 Barcelona

COUNTRY: Spain

POSTAL CODE (ZIP): none

(ii) TITLE OF INVENTION: CONOTOXINS I (iii) NUMBER OF SEQUENCES: 13

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO)

(v) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/084,848

(B) FILING DATE: June 29, 1993

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Gly Cys Cys Gly Ser Tyr Pro Asn Ala Ala Cys His Pro Cys Ser Cys 1 5 10 15

Lys Asp Arg Xaa Ser Tyr Cys Gly Gin 20 25

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Glu Lys Ser Leu Val Pro Ser Val lie Thr Thr Cys Cys Gly Tyr Asp 1 5 10 15

Xaa Gly Thr Met Cys Xaa Xaa Cys Arg Cys Thr Asn Ser Cys 20 25 30

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO: 3 :

Cys Cys Gly Val Xaa Asn Ala Ala Cys Pro Xaa Cys Val Cys Asn Lys 1 5 10 15

Thr Cys Gly

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Gly Cys Cys Gly Ser Tyr Xaa Asn Ala Ala Cys His Xaa Cys Ser Cys 1 5 10 15

Lys Asp Arg Xaa Ser Tyr Cys Gly Gin 20 25

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Gly Cys Cys Gly Ser Tyr Xaa Asn Ala Ala Cys His Pro Cys Ser Cys 1 5 10 15

Lys Asp Arg Xaa Ser Tyr Cys Gly Gin 20 25

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 6 :

Cys Cys Gly Val Xaa Asn Ala Ala Cys His Xaa Cys Val Cys Lys Asn 1 5 10 15

Thr Cys

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 46 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Gly Xaa Ser Phe Cys Lys Ala Asp Glu Lys Xaa Cys Glu Tyr His Ala 1 5 10 15

Asp Cys Cys Asn Cys Cys Leu Ser Gly lie Cys Ala Xaa Ser Thr Asn 20 25 30

Trp lie Leu Pro Gly Cys Ser Thr Ser Ser Phe Phe Lys lie 35 40 45

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Gly Cys Cys Ser His Pro Ala Cys Ser Gly Lys Tyr Gin Xaa Tyr Cys 1 5 10 15

Arg Xaa Ser

(2) INFORMATION FOR SEQ ID NO:9;

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

His Xaa Xaa Cys Cys Leu Tyr Gly Lys Cys Arg Arg Tyr Xaa Gly Cys 1 5 10 15

Ser Ser Ala Ser Cys Cys Gin 20

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Cys Lys Thr Tyr Ser Lys Tyr Cys Xaa Ala Asp Ser Xaa Cys Cys Thr 1 5 10 15

Xaa Gin Cys Val Arg Ser Tyr Cys Thr Leu Phe 20 25

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:

Ser Thr Ser Cys Met Glu Ala Gly Ser Tyr Cys Gly Ser Thr Thr Arg 1 5 10 15 lie Cyε Cys Gly Tyr Cys Ala Tyr Phe Gly Lys Lys Cys lie Asp Tyr 20 25 30

Pro Ser Asn 35

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

Gly Glu Xaa Xaa Val Ala Lys Met Ala Ala Xaa Leu Ala Arg Xaa Asn 1 5 10 15 lie Ala Lys Gly Cys Lys Val Asn Cys Tyr Pro 20 25

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Glu Ser Glu Glu Gly Gly Ser Asn Ala Thr Lys Lys Pro Tyr lie Leu 1 5 10 15